Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed primarily in subanatomical portions of the brain, T lymphocytes and postmeiotic male germ cells. It is present in the nucleus of cells in which it is expressed (Jensen et al, Proc. Natl. Acad. Sci. USA 88:2850 (1991)). CaMKIV phosphorylates and activates the cyclic AMP response element binding proteins CREB and CREMτ in a manner analogous to protein kinase A (Matthews et al, Mol. Cell. Biol. 14:6107 (1994); Sun et al, Genes Dev. 8:2527 (1994); Enslen et al, J. Biol. Chem. 269:15320 (1994)).
In the absence of Ca2+/calmodulin, CaMKIV is inactive. Activation requires three events: i) binding of Ca2+/calmodulin; ii) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca2+/calmodulin-dependent; and iii) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition.
CaMKIV has previously been implicated in mediating cytokine gene transcription during T cell activation using a line of transgenic mice that overexpress a catalytically inactive form of CaMKIV (Anderson et al, Mol. Endo. 11:725-737 (1997)). Driven by the murine Ick promoter, the mutant CaMKIV was expressed only in developing thymoctes and thus, the differentiation of mature T cells could not be addressed in these mice. High level expression of CaMKIV is restricted to only a few cell types, including the CD4+ T cell, where it has been localized to the nucleus in a tight 1:1 stoichiometric association with protein phosphatase 2A (Hanissian et al, J. Biol. Chem. 268:20055-20063 (1993), Westphal et al, Science 280:1258-1261 (1998)).
The specific role of CaMKIV in T cell activation is described in U.S. application Ser. No. 09/033,715, as is the importance of CaMKIV as a target for immunosuppressive agents. A method of identifying immunosuppressive agents that target CaMKIV is also provided in U.S. application Ser. No. 09/033,715. Agents identified using the screen provided there are T cell specific and thus substantially free of adverse side effects.
The studies described herein relate, at least in part, to the role of CaMKIV in CD4+ T cell differentiation. Naïve CD4+ T cells can differentiate along different pathways in response to stimulation by MHC/antigen complex (see FIG. 1). The most well characterized T helper subsets are referred to as Th1 and Th2 which differ in the types of cytokines they secrete. The different sets of cytokines in turn mediate different effector functions. Th1 cells secrete IL-2, INF-γ, and TNF-β. IL-2 functions as the Th1 cell autocrine growth factor and promotes the differentiation of CD8+ T cells into cytolytic T cells. IFN-γ stimulates macrophages to kill intracellular microbes and activates the production of IgG which is the principle immunoglobulin involved in opsonization and phagocytosis. The TNF-β secreted by Th1 cells recruits and activates inflammatory leukocytes. Collectively, these are mechanisms used by Th1 cells to promote elimination of intracellular microbes. In contrast, Th2 cells express IL-4, IL-5 and IL-13. IL-4 functions as the Th2 autocrine growth factor. This cytokine is also required for immunoglobulin switching to the IgE isotype and is the primary activator of mast cells. IL-5 stimulates eosinophils to kill parasites. IL-13 is required for generation of allergic asthma. Th2 cells thus function to eliminate parasites and are the mediators of allergic responses.
The mechanisms determining the pathway by which an individual, naïve T cell proceeds are unclear, but the process is strongly influenced by the context in which T cell stimulation occurs. Once a Th1 or Th2 population begins to become established, the cytokines it produces inhibit the alternative pathway (FIG. 1) to further promote dominance of a single cell type. In vivo, one or the other differentiated cell type generally comes to dominate during the course of an immune response to profoundly determine the outcome of the response.
The present invention provided methods of identifying agonists (activators) and antagonists (inhibitors) of CaMKIV and to methods of using compounds so identified in a variety of therapeutic settings.